Glycerophospholipids, the main component of biological membranes, contain a glycerol core with fatty acids attached as R groups at the sn-1 position and sn-2 position, and a polar head group joined at the sn-3 position via a phosphodiester bond. The specific polar head group (e.g., phosphatidic acid, chloline, ethanolamine, glycerol, inositol, serine, cardiolipin) determines the name given to a particular glycerophospholipid, thus resulting in phosphatidylcholines [“PC”], phosphatidylethanolamines [“PE”], phosphatidylglycerols [“PG”], phosphatidylinositols [“PI”], phosphatidylserines [“PS”] and cardiolipins [“CL”]. Glycerophospholipids possess tremendous diversity, not only resulting from variable phosphoryl head groups, but also as a result of differing chain lengths and degrees of saturation of their fatty acids. Generally, saturated and monounsaturated fatty acids are esterified at the sn-1 position, while polyunsaturated fatty acids are esterified at the sn-2 position.
Glycerophospholipid biosynthesis is complex. Table 1 below summarizes the steps in the de novo pathway, originally described by Kennedy and Weiss (J. Biol. Chem., 222:193-214 (1956)):
TABLE 1General Reactions Of de Novo Glycerophospholipid Biosynthesissn-Glycerol-3-PhosphateGlycerol-3-phosphate acyltransferase→ Lysophosphatidic Acid(GPAT) [E.C. 2.3.1.15] esterifies 1st acyl-(1-acyl-sn-glycerol 3-CoA to sn-1 position of sn-glycerolphosphate or “LPA”)3-phosphateLPA → Phosphatidic AcidLysophosphatidic acid acyltransferase(1,2-diacylglycerol(LPAAT) [E.C. 2.3.1.51] esterifies 2nd acyl-phosphate or “PA”)CoA to sn-2 position of LPAPA → 1,2-DiacylglycerolPhosphatidic acid phosphatase [E.C. 3.1.3.4](“DAG”)removes a phosphate from PA; DAG cansubsequently be converted to PC, PE orTAG (TAG synthesis requires either adiacylglycerol acyltransferase (DGAT)[E.C. 2.3.1.20] or a phospholipid:diacyl-glycerol acyltransferase (PDAT)Or[E.C. 2.3.1.158])PA → Cytidine DiphosphateCDP-diacylglycerol synthase [EC 2.7.7.41]Diacylglycerol (“CDP-DG”)causes condensation of PA and cytidinetriphosphate, with elimination of pyrophosphate; CDP-DG can subsequentlybe converted to PI, PS, PG or CL
Following their de novo synthesis, glycerophospholipids can undergo rapid turnover of the fatty acyl composition at the sn-2 position. This “remodeling”, or “acyl editing”, is important for membrane structure and function, biological response to stress conditions, and manipulation of fatty acid composition and quantity in biotechnological applications. Specifically, the remodeling has been attributed to deacylation of the glycerophospholipid and subsequent reacylation of the resulting lysophospholipid.
In the Lands' cycle (Lands, W. E., J. Biol. Chem., 231:883-888 (1958)), remodeling occurs through the concerted action of: 1) a phospholipase, such as phospholipase A2, that releases fatty acids from the sn-2 position of phosphatidylcholine; and, 2) acyl-CoA:lysophospholipid acyltransferases [“LPLATs”], such as lysophosphatidylcholine acyltransferase [“LPCAT”] that reacylates the lysophosphatidylcholine [“LPC”] at the sn-2 position. Other glycerophospholipids can also be involved in the remodeling with their respective lysophospholipid acyltransferase activity, including LPLAT enzymes having lysophosphatidylethanolamine acyltransferase [“LPEAT”]activity, lysophosphatidylserine acyltransferase [“LPSAT”] activity, lysophosphatidylglycerol acyltransferase [“LPGAT”] activity and lysophosphatidylinositol acyltransferase [“LPIAT”] activity. In all cases, LPLATs are responsible for removing acyl-CoA fatty acids from the cellular acyl-CoA pool and acylating various lysophospholipid substrates at the sn-2 position in the phospholipid pool. Finally, LPLATs also include LPAAT enzymes that are involved in the de novo biosynthesis of PA from LPA. LPCAT activity is associated with two structurally distinct protein families, wherein one belongs to the LPAAT family of proteins and the other belongs to the membrane bound O-acyltransferase [“MBOAT”] family of proteins.
In other cases, this sn-2 position remodeling has been attributed to the forward and reverse reactions of enzymes having LPCAT activity (Stymne S. and A. K. Stobart, Biochem J., 223(2):305-314 (1984)).
Several recent reviews by Shindou et al. provide an overview of glycerophospholipid biosynthesis and the role of LPLATs (J. Biol. Chem., 284(1):1-5 (2009); J. Lipid Res., 50:S46-S51 (2009)). Numerous LPLATs have been reported in public and patent literature, based on a variety of conserved motifs.
The effect of LPLATs on polyunsaturated fatty acid [“PUFA”] production has also been contemplated, since fatty acid biosynthesis requires rapid exchange of acyl groups between the acyl-CoA pool and the phospholipid pool. Specifically, desaturations occur mainly at the sn-2 position of phospholipids, while elongation occurs in the acyl-CoA pool. For example, Intl. App. Pub. No. WO 2004/076617 describes the isolation of an LPCAT from Caenorhabditis elegans (clone T06E8.1) and reports increase in the efficiency of Δ6 desaturation and Δ6 elongation, as well as an increase in biosynthesis of the long-chain PUFAs eicosadienoic acid [“EDA”; 20:2] and eicosatetraenoic acid [“ETA”; 20:4], respectively, when the LPCAT was expressed in an engineered strain of Saccharomyces cerevisiae that was fed exogenous 18:2 or α-linolenic [“ALA”; 18:3] fatty acids, respectively.
Furthermore, Example 16 of Intl. App. Pub. No. WO 2004/087902 describes the isolation of Mortierella alpina LPAAT-like proteins (encoded by the proteins of SEQ ID NO:93 and SEQ ID NO:95, having 417 amino acids in length or 389 amino acids in length, respectively) that are identical except for an N-terminal extension of 28 amino acid residues in SEQ ID NO:93. Intl. App. Pub. No. WO 2004/087902 also reports expression of one of these proteins using similar methods to those of Intl. App. Pub. No. WO 2004/076617, which results in similar improvements in EDA and ETA biosynthesis.
Both Intl. App. Publications No. WO 2004/076617 and No. WO 2004/087902 teach that the improvements in EDA and ETA biosynthesis are due to reversible LPCAT activity in some LPAAT-like proteins, although not all LPAAT-like proteins have LPCAT activity. They do not teach that LPCAT expression would result in the improvements in strains that do not require exogenous feeding of fatty acid substrates or in microbial species other than Saccharomyces cerevisiae. They also do not teach that LPCAT expression in engineered microbes results in increased production of high LC-PUFAs other than EDA and ETA, such as ARA, EPA and DHA, or that LPCAT expression can result in improvement in alternate desaturation reactions, other than Δ6 desaturation. Neither reference teaches the effect of the LPCAT or LPAAT-like proteins on either Δ6 elongation without exogenous feeding of fatty acids or on Δ4 desaturation.
Numerous other references generally describe benefits of co-expressing LPLATs with PUFA biosynthetic genes, to increase the amount of a desired fatty acid in the oil of a transgenic organism, increase total oil content or selectively increase the content of desired fatty acids (e.g., Intl. App. Pubublications No. WO 2004/087902, No. WO 2006/069936, No. WO 2006/052870, No. WO 2009/001315, No. WO 2009/014140).
Despite the work describe above, to date no one has studied the effect of LPAATs and LPCATs in an oleaginous organism engineered for high-level production of LC-PUFAs other than EDA and ETA, such as eicosapentaenoic acid [“EPA”; cis-5,8,11,14,17-eicosapentaenoic acid] and/or docosahexaenoic acid [“DHA”; cis-4,7,10,13,16,19-docosahexaenoic acid] and for improved C18 to C20 elongation conversion efficiency, and/or improved Δ4 desaturation conversion efficiency without exogenously feeding fatty acids.